Although genomics has advanced the genetic basis of biological processes, it has significant limitations in understanding the complete biological process. It is therefore now generally accepted that the biological process cannot be completely defined or understood solely by the genetic sequence or MRNA transcripts. A complete understanding of the biological process must entail the structure and dynamics of proteins. The study of the protein complement of the genome is commonly referred to as Proteomics.
The function of products encoded by identified genes and especially by partial cDNA sequences are frequently unknown as is information about post-translational modifications (such as glycosylation and phosphorylation) that can profoundly influence their biochemical properties. Protein expression is often subject to post-translational control, so that the cellular level of an mRNA does not necessarily correlate with the expression level of its gene product. Automated techniques for random sequencing of nucleic acids involve the analysis of large numbers of nucleic acid molecules prior to determining which, if any, show indications of scientific significance. For these reasons, there is a need to supplement genomic data by studying the patterns of protein expression, and of post-translational modification, in a biological or disease process through direct analysis of proteins and protein digests.
Technical constraints, however, have heretofore limited the automated, cost-effective, reproducible, systematic analysis of proteins and other biomolecules present in biological samples. Analyzing biomolecules such as proteins needs to be substantially automated to avoid time consuming, labor intensive, expensive, and inefficient detection, imaging, purification and analysis.
One of the most robust methods of analysis of biological samples is Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS). Sample analysis by MALDI is generally known to be a simple technique with high sensitivity without the use of chromatographic separation prior to analysis. MALDI currently allows precise measurements of masses of peptides from protein digests having molecular weights ranging from about 500 to 5000 daltons, as well as intact proteins up to 100,000 Da. While single protein digests can be analyzed by MALDI-TOF/MS to generate peptide mass fingerprints (PMF) that identify the protein when a set of proteins from a sample are mixed together, detection of such a set by PMF provides more challenges for identification by PMF. The protein digests can also be analyzed in MALDI-MS/MS instruments that allow for the fragmentation of individual peptides and subsequent sequencing and identification of the protein from which that peptide originated.
The present detection limits with mass spectrometry, especially MALDI, depend on getting a sample concentrated and onto a small target area. Unfortunately, present methods of concentrating a sample onto a MALDI target include the use of laboratory tools such as pipette tips containing resin attached at their very tip occupying about 0.5 μl volume, such as ZipTip™ (Millipore, Bedford, Mass.). This method of transfer using ZipTips suffers from a diffential loss of sample material that is adsorbed to the surface of the resin bed and consequently this method is not practical for protein digests where poor recovery is an issue.
Analysis of biological samples by MALDI is currently a process that is primarily an off-line technique that requires manual or automated preparation of a sample mixed with a matrix material. This off-line preparation requires a great deal of expertise and a corresponding significant amount of time. The process can be automated by use of liquid handling robots and ZipTips or by use of a system to desolvate the eluent from some desalting column. For the latter case prior attempts to automate this off-line process have been met with technical constraints.
In U.S. Pat. No. 6,175,112 to Karger et al., a universal interface for continuous on-line liquid sample introduction directly to a time-of-flight mass spectrometer is described. Unfortunately this attempt to automate and therefore increase the throughput and utility of MALDI-TOF mass spectrometry suffers from several deficiencies. The eluent flow rate of Karger is limited to less than about 300 nanoliters per minute and therefore larger scale chromatography, such as capillary liquid chromatography, cannot be accommodated without a post column split. Further, the liquid junction of the fused silica tip is subject to plugging and the silica tip requires frequent replacement. Additionally, the Karger configuration requires a separate auxiliary pump for matrix flow into the liquid junction and vacuum chamber.